Gene network activity in cultivated primary hepatocytes is highly similar to diseased mammalian liver tissue

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Abstract

It is well known that isolation and cultivation of primary hepatocytes cause major gene expression alterations. In the present genome-wide, time-resolved study of cultivated human and mouse hepatocytes, we made the observation that expression changes in culture strongly resemble alterations in liver diseases. Hepatocytes of both species were cultivated in collagen sandwich and in monolayer conditions. Genome-wide data were also obtained from human NAFLD, cirrhosis, HCC and hepatitis B virus-infected tissue as well as mouse livers after partial hepatectomy, CCl ₄ intoxication, obesity, HCC and LPS. A strong similarity between cultivation and disease-induced expression alterations was observed. For example, expression changes in hepatocytes induced by 1-day cultivation and 1-day CCl ₄ exposure in vivo correlated with R = 0.615 ( p < 0.001). Interspecies comparison identified predominantly similar responses in human and mouse hepatocytes but also a set of genes that responded differently. Unsupervised clustering of altered genes identified three main clusters: (1) downregulated genes corresponding to mature liver functions, (2) upregulation of an inflammation/RNA processing cluster and (3) upregulated migration/cell cycle-associated genes. Gene regulatory network analysis highlights overrepresented and deregulated HNF4 and CAR (Cluster 1), Krüppel-like factors MafF and ELK1 (Cluster 2) as well as ETF (Cluster 3) among the interspecies conserved key regulators of expression changes. Interventions ameliorating but not abrogating cultivation-induced responses include removal of non-parenchymal cells, generation of the hepatocytes’ own matrix in spheroids, supplementation with bile salts and siRNA-mediated suppression of key transcription factors. In conclusion, this study shows that gene regulatory network alterations of cultivated hepatocytes resemble those of inflammatory liver diseases and should therefore be considered and exploited as disease models.

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Most cited references 20

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Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron.

Trude Flo, Kelly D. Smith, Shintaro Sato … (2004)

Although iron is required to sustain life, its free concentration and metabolism have to be tightly regulated. This is achieved through a variety of iron-binding proteins including transferrin and ferritin. During infection, bacteria acquire much of their iron from the host by synthesizing siderophores that scavenge iron and transport it into the pathogen. We recently demonstrated that enterochelin, a bacterial catecholate siderophore, binds to the host protein lipocalin 2 (ref. 5). Here, we show that this event is pivotal in the innate immune response to bacterial infection. Upon encountering invading bacteria the Toll-like receptors on immune cells stimulate the transcription, translation and secretion of lipocalin 2; secreted lipocalin 2 then limits bacterial growth by sequestrating the iron-laden siderophore. Our finding represents a new component of the innate immune system and the acute phase response to infection.

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CellNet: network biology applied to stem cell engineering.

Patrick Cahan, Hu Li, Samantha A. Morris … (2014)

Somatic cell reprogramming, directed differentiation of pluripotent stem cells, and direct conversions between differentiated cell lineages represent powerful approaches to engineer cells for research and regenerative medicine. We have developed CellNet, a network biology platform that more accurately assesses the fidelity of cellular engineering than existing methodologies and generates hypotheses for improving cell derivations. Analyzing expression data from 56 published reports, we found that cells derived via directed differentiation more closely resemble their in vivo counterparts than products of direct conversion, as reflected by the establishment of target cell-type gene regulatory networks (GRNs). Furthermore, we discovered that directly converted cells fail to adequately silence expression programs of the starting population and that the establishment of unintended GRNs is common to virtually every cellular engineering paradigm. CellNet provides a platform for quantifying how closely engineered cell populations resemble their target cell type and a rational strategy to guide enhanced cellular engineering.

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Gene expression profiles during hepatic stellate cell activation in culture and in vivo.

Johannes Kluwe, Yonghui Zhang, Ekihiro Seki … (2007)

Following hepatic injury, hepatic stellate cells (HSCs) transdifferentiate to become extracellular matrix-producing myofibroblasts and to promote hepatic fibrogenesis. In this study, we determine gene expression changes in 3 different models of HSC activation and investigate whether HSC culture activation reproduces gene expression changes of HSC in vivo activation. HSCs were isolated by density centrifugation and magnetic antibody cell sorting from normal mice, CCl(4)-treated mice, and mice that underwent bile duct ligation (BDL). Gene expression was analyzed by microarray and confirmed by polymerase chain reaction and Western blot analysis. Two thousand seventy-three probe sets were differentially expressed in at least 1 of 3 models of HSC activation, including novel genes that encode proinflammatory and antiapoptotic mediators; transcription factors; cell surface receptors; and cytoskeleton components such as CXCL14, survivin, septin 4, osteopontin, PRX1, LMCD1, GPR91, leiomodin, and anillin. BDL- and CCl(4)-activated HSCs showed highly correlated gene expression patterns, whereas culture activation only partially reproduced the gene expression changes observed during BDL- and CCl(4)-induced activation. Coculture with Kupffer cells or lipopolysaccharide treatment during culture activation shifted the expression of most examined genes toward the pattern observed during in vivo activation, suggesting a role for these factors in the microenvironment that drives HSC activation. The almost identical HSC gene expression patterns after BDL or CCl(4) treatment indicate that HSCs exert similar functions in different types of liver injury. Because culture activation does not properly regulate gene expression in HSCs, in vivo activation should be considered the gold standard for the study of HSC biology.

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Author and article information

Contributors

Patricio Godoy: ++49-0231-108-4370 , godoy@ifado.de

Jan G. Hengstler: hengstler@ifado.de

Journal

Journal ID (nlm-ta): Arch Toxicol

Journal ID (iso-abbrev): Arch. Toxicol

Title: Archives of Toxicology

Publisher: Springer Berlin Heidelberg (Berlin/Heidelberg )

ISSN (Print): 0340-5761

ISSN (Electronic): 1432-0738

Publication date (Electronic): 23 June 2016

Publication date PMC-release: 23 June 2016

Publication date (Print): 2016

Volume: 90

Issue: 10

Pages: 2513-2529

Affiliations

[1 ]IfADo-Leibniz Research Centre for Working Environment and Human Factors, Technical University of Dortmund, Ardeystrasse 67, 44139 Dortmund, Germany

[2 ]Leibniz Institute for Natural Product Research and Infection Biology eV-Hans-Knöll Institute, Jena, Germany

[3 ]Molecular Alcohol Research in Gastroenterology, Department of Medicine II, Faculty of Medicine at Mannheim, University of Heidelberg, Mannheim, Germany

[4 ]BG Trauma Center, Siegfrid Weller Insitut, Eberhard Karls University Tübingen, Tübingen, Germany

[5 ]Center for Liver Cell Research, Department of General, Visceral, Transplantation, Vascular and Thorax Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich, Germany

[6 ]Department of General, Visceral and Transplantation Surgery, Charité University Medicine Berlin, Berlin, Germany

[7 ]Center for Liver Cell Research, University Children Hospital (KUNO), Regensburg University Hospital, Regensburg, Germany

[8 ]Institute of Pathology, Friedrich-Schiller-University of Jena, Jena, Germany

[9 ]Experimental Transplantation Surgery, Department of General, Visceral and Vascular Surgery, University Hospital Jena, Friedrich-Schiller-University of Jena, Jena, Germany

[10 ]Institute of Biochemistry, Faculty of Medicine, University of Leipzig, Leipzig, Germany

[11 ]Institute of Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany

[12 ]InSphero AG, Wagistrasse 27, 8952 Schlieren, Switzerland

[13 ]Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, University of Tuebingen, Auerbachstrasse 112, 70376 Stuttgart, Germany

[14 ]Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Dortmund, Germany

[15 ]Institute of Pathology, Charité-Universitätsmedizin Berlin, Berlin, Germany

[16 ]Department of Forensic and Veterinary Toxicology, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt

[17 ]Facultad de Ciencias Biológicas, Departamento de Fisiología, Universidad de Concepción, Concepción, Chile

[18 ]Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO USA

Article

Publisher ID: 1761

DOI: 10.1007/s00204-016-1761-4

PMC ID: 5043005

PubMed ID: 27339419

SO-VID: ccaf1c05-ba98-4408-bc0b-6599f841e274

License:

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

History

Date received : 23 March 2016

Date accepted : 13 June 2016

Funding

Funded by: FundRef http://dx.doi.org/10.13039/501100004963, Seventh Framework Programme;

Award ID: 267038

Award ID: 266838

Award Recipient : Jan G. Hengstler

Funded by: FundRef http://dx.doi.org/10.13039/501100002347, Bundesministerium für Bildung und Forschung;

Award ID: 0313854

Award ID: 0315753

Award Recipient : Thomas S. Weiss Jan G. Hengstler

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ScienceOpen disciplines: Toxicology

Keywords: gene arrays,bioinformatics,inflammation,metabolism,differentiation

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ScienceOpen disciplines: Toxicology

Keywords: gene arrays, bioinformatics, inflammation, metabolism, differentiation

Gene network activity in cultivated primary hepatocytes is highly similar to diseased mammalian liver tissue

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Journal of the Netherlands Society of Toxicology

Most cited references 20

Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron.

CellNet: network biology applied to stem cell engineering.

Gene expression profiles during hepatic stellate cell activation in culture and in vivo.

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